Antisense modulation of lysophospholipase I expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of Lysophospholipase I. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Lysophospholipase I. Methods of using these compounds for modulation of Lysophospholipase I expression and for treatment of diseases associated with expression of Lysophospholipase I are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of Lysophospholipase I. In particular, this inventionrelates to antisense compounds, particularly oligonucleotides,specifically hybridizable with nucleic acids encoding LysophospholipaseI. Such oligonucleotides have been shown to modulate the expression ofLysophospholipase I.

BACKGROUND OF THE INVENTION

Phospholipids, considered the building blocks of biological membranes,function not only as structural barriers for the cell but are vitalconstituents, acting as second messengers, of signal transductionpathways. It has been demonstrated that oxidized phospholipids play akey role in the development of certain diseases, and therefore thestudy-of phospholipid metabolism has become a field of intense study.

Lysophospholipids (LysoPLs) are intermediates of phospholipidmetabolism. These lipids consist of one long hydrophobic acyl chain andone large hydrophilic polar head group, making them amphipathic (havingboth positive and negatively charged characteristics). This qualitygives the lysophospholipids surfactant and detergent properties and thustheir levels must be strictly regulated for proper cell function andsurvival. Increased levels of lysophospholipids could result indisruption of membrane structure and possibly cell lysis. Consequently,increased levels of lysophospholipids have been associated with avariety of disease processes (Wang and Dennis, Biochim. Biophys. Acta.,1999, 1439, 1-16).

Regulation of lysophospholipid levels is controlled by a family ofenzymes known as lysophospholipases (LysoPLAs). These enzymes controlthe levels of lysophospholipids through hydrolysis and are the majorpathway by which lysophospholipids are degraded (Wang and Dennis,Biochim. Biophys. Acta., 1999, 1439, 1-16).

Lysophospholipases are divided into low and high molecular weightisoforms with varying substrate specificity and pH requirements. Thehigh molecular weight isoforms act as hydrolases and transacylases whilethe low molecular weight isoforms act as hydrolases. Many species havetwo low molecular weight forms, lysophospholipase A I (LysoPLA I) andlysophospholipase A II (LysoPLA II).

Lysophospholipase I (also known as LPL1, LYPLA1 and LysoPLA I) was firstsequenced and cloned from a rat liver cDNA library using antibodytechniques. It was subsequently cloned from the mouse and human (Wang etal., J. Biol. Chem., 1997, 272, 12723-12729; Wang et al., Biochim.Biophys. Acta., 1999, 1437, 157-169).

Disclosed in U.S. Pat. Nos. 5,858,756, 5,965,423 and the PCT publicationWO 98/49319 are the polynucleotide and polypeptide sequence of the humanlysophospholipase A I (NHLP) as well as vectors, host cells and methodsfor expressing the enzyme. Isolated polynucleotides completelycomplementary to a polynucleotide encoding NHLP are also disclosed.Further disclosed are antibodies to the enzyme, agonists and antagonistsof the polypeptide as well as a purified polynucleotide which hybridizesunder stringent conditions to the polynucleotide which encodeslysophospholipase I (Hillman et al., 1998; Hillman et al., 1999).

Tissue distribution studies by Northern and Western blots indicate thatthe lysophospholipase A I mRNA and protein are widely distributed in alltissues examined (Wang et al., Biochim. Biophys. Acta., 1999, 1437,157-169). In the human adult tissues, the highest expression is seen inthe heart, placenta and skeletal muscle while fetal tissues demonstrateda more uniform pattern of expression (Wang et al., Biochim. Biophys.Acta., 1999, 1437, 157-169).

Lysophospholipase I has also been shown to mediate other functionswithin the cell including G-protein signal transduction. It has beenshown to remove the palmitate group from G-protein alpha subunits(Duncan and Gilman, J. Biol. Chem., 1998, 273, 15830-15837).

The pharmacological modulation of lysophospholipase I activity and/orexpression may therefore be an appropriate point of therapeuticintervention in pathological conditions involving deregulatedphospholipid metabolism.

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of lysophospholipase I. The most potent inhibitorof lysophospholipase I activity is MAFP (methyl arachidonylfluorophosphonate) which irreversibly inhibits enzyme activity (Wang etal., Biochim. Biophys. Acta., 1999, 1437, 157-169). Consequently, thereremains a long felt need for additional agents capable of effectivelyinhibiting lysophospholipase I function.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and may therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications for the modulation of lysophospholipase I expression.

The present invention provides compositions and methods for modulatinglysophospholipase I expression.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, particularlyoligonucleotides, which are targeted to a nucleic acid encodingLysophospholipase I, and which modulate the expression ofLysophospholipase I. Pharmaceutical and other compositions comprisingthe antisense compounds of the invention are also provided. Furtherprovided are methods of modulating the expression of Lysophospholipase Iin cells or tissues comprising contacting said cells or tissues with oneor more of the antisense compounds or compositions of the invention.Further provided are methods of treating an animal, particularly ahuman, suspected of having or being prone to a disease or conditionassociated with expression of Lysophospholipase I by administering atherapeutically or prophylactically effective amount of one or more ofthe antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding Lysophospholipase I, ultimatelymodulating the amount of Lysophospholipase I produced. This isaccomplished by providing antisense compounds which specificallyhybridize with one or more nucleic acids encoding Lysophospholipase I.As used herein, the terms “target nucleic acid” and “nucleic acidencoding Lysophospholipase I” encompass DNA encoding LysophospholipaseI, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and alsocDNA derived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression ofLysophospholipase I. In the context of the present invention,“modulation” means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. In the context of the presentinvention, inhibition is the preferred form of modulation of geneexpression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding Lysophospholipase I. The targeting process alsoincludes determination of a site or sites within this gene for theantisense interaction to occur such that the desired effect, e.g.,detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding Lysophospholipase I, regardless of the sequence(s)of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′ UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′ UTR), known in the art to refer tothe portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA or correspondingnucleotides on the gene. The 5′ cap of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap The 5′ cap region may also be apreferred target region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “Introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans. In the context of this invention, the term“oligonucleotide” refers to an oligomer or polymer of ribonucleic acid(RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This termincludes oligonucleotides composed of naturally-occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally-occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for nucleic acidtarget and increased stability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 30 nucleobases (i.e.from about 8 to about 30 linked nucleosides). Particularly preferredantisense compounds are antisense oligonucleotides, even more preferablythose comprising from about 12 to about 25 nucleobases. As is known inthe art, a nucleoside is a base-sugar combination. The base portion ofthe nucleoside is normally a heterocyclic base. The two most commonclasses of such heterocyclic bases are the purines and the pyrimidines.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Informing oligonucleotides, the phosphate groups covalently link adjacentnucleosides to one another to form a linear polymeric compound. In turnthe respective ends of this linear polymeric structure can be furtherjoined to form a circular structure, however, open linear structures aregenerally preferred. Within the oligonucleotide structure, the phosphategroups are commonly referred to as forming the internucleoside backboneof the oligonucleotide. The normal linkage or backbone of RNA and DNA isa 3′ to 5′ phosphodiester linkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos.: 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.:5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH2— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′—O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′—O—CH₃),2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂) and 2′ -fluoro (2′—F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4 thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 andO-substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference, and U.S. Pat. No. 5,750,692, which iscommonly owned with the instant application and also herein incorporatedby reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for F example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of Lysophospholipase I is treated by administering antisensecompounds in accordance with this invention. The compounds of theinvention can be utilized in pharmaceutical compositions by adding aneffective amount of an antisense compound to a suitable pharmaceuticallyacceptable diluent or carrier. Use of the antisense compounds andmethods of the invention may also be useful prophylactically, e.g., toprevent or delay infection, inflammation or tumor formation, forexample.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingLysophospholipase I, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encodingLysophospholipase I can be detected by means known in the art. Suchmeans may include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofLysophospholipase I in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets;capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and-contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system. ATherefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p.92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9 th Ed., Hardman et al. Eds., McGraw-Hill, New York,1996, pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18 thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 619, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). Other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham MA or GlenResearch, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2′-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2′-Fluoro amidites

2′-Fluorodeoxyadenosine amidites

2′-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-O-(2-Methoxyethyl) modified amidites

2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro [1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, TLC showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO, and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1 M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1 M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′—-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) nucleoside amidites

2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosin-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine ribosidemay be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced byN-hydroxyphthalimide via a Mitsunobu reaction, and the protectednucleoside may phosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′—-O-dimethylaminoethoxyethyl, i.e., 2′—O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowlyadded to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-, 2-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyluridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2 Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No., 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3 Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic& MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[2′O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy-]-[2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleo-sides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing 6 cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, Ribonucleaseprotection assays, or RT-PCR.

T-24 cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5 A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

HepG2 cells:

The human hepatoblastoma cell line HepG2 was obtained from the AmericanType Culure Collection (Manassas, Va.). HepG2 cells were routinelycultured in Eagle's MEM supplemented with 10% fetal calf serum,non-essential amino acids, and 1 mM sodium pyruvate (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

b.END cells:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 3000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with antisense compounds:

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration of apositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10 Analysis of Oligonucleotide Inhibition of Lysophospholipase Iexpression

Antisense modulation of Lysophospholipase I expression can be assayed ina variety of ways known in the art. For example, Lysophospholipase ImRNA levels can be quantitated by, e.g., Northern blot analysis,competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR).Real-time quantitative PCR is presently preferred. RNA analysis can beperformed on total cellular RNA or poly(A)+ mRNA. Methods of RNAisolation are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis isroutine in the art and is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, JohnWiley & Sons, Inc., 1996. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISM™7700 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed as multiplexable.Other methods of PCR are also known in the art.

Protein levels of Lysophospholipase I can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to Lysophospholipase I can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional antibody generation methods. Methods for preparation ofpolyclonal antisera are taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9,John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons,Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11 Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

Total mRNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-time Quantitative PCR Analysis of Lysophospholipase ImRNA Levels

Quantitation of Lysophospholipase I mRNA levels was determined byreal-time quantitative PCR using the ABI PRISM™ 7700 Sequence DetectionSystem (PE-Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR, in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., JOE, FAM, or VIC, obtained from either OperonTechnologies Inc., Alameda, Calif. or PE-Applied Biosystems, FosterCity, Calif.) is attached to the 5′ end of the probe and a quencher dye(e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda,Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the3′ end of the probe. When the probe and dyes are intact, reporter dyeemission is quenched by the proximity of the 3′ quencher dye. Duringamplification, annealing of the probe to the target sequence creates asubstrate that can be cleaved by the 5′-exonuclease activity of Taqpolymerase. During the extension phase of the PCR amplification cycle,cleavage of the probe by Taq polymerase releases the reporter dye fromthe remainder of the probe (and hence from the quencher moiety) and asequence-specific fluorescent signal is generated. With each cycle,additional reporter dye molecules are cleaved from their respectiveprobes, and the fluorescence intensity is monitored at regular intervalsby laser optics built into the ABI PRISM™ 7700 Sequence DetectionSystem. In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1×TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension).

Probes and primers to human Lysophospholipase I were designed tohybridize to a human Lysophospholipase I sequence, using publishedsequence information (GenBank accession number AF081281, incorporatedherein as SEQ ID NO:3). For human Lysophospholipase I the PCR primerswere: forward primer: TCCAGCCAATGTGACCTTTAAA (SEQ ID NO: 4) reverseprimer: AATGAATTGCTTGACATCCATCA (SEQ ID NO: 5) and the PCR probe was:FAM-CCTATGAAGGTATGATGCACAGTTCGTGTCAA-TAMRA (SEQ ID NO: 6) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye. For human GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)

reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probewas: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where JOE(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye.

Probes and primers to mouse Lysophospholipase I were designed tohybridize to a mouse Lysophospholipase I sequence, using publishedsequence information (GenBank accession number U89352, incorporatedherein as SEQ ID NO:10). For mouse Lysophospholipase I the PCR primerswere: forward primer: GGCTATGCCTTCTTGGTTTGATA (SEQ ID NO:11) reverseprimer: TGCCTGTTTAATTCCAGATTCATC (SEQ ID NO: 12) and the PCR probe was:FAM-CGTTGGACTTTCACCAGATTCCCAGG-TAMRA (SEQ ID NO: 13) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye. For mouse GAPDH the PCR primers were:

forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14)

reverse primer: GGGTCTCGCTCCTGGAAGCT (SEQ ID NO: 15) and the PCR probewas: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 16) whereJOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescentreporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) isthe quencher dye.

Example 14 Northern Blot Analysis of Lysophospholipase I mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human Lysophospholipase I, a human Lysophospholipase Ispecific probe was prepared by PCR using the forward primerTCCAGCCAATGTGACCTTTAAA (SEQ ID NO: 4) and the reverse primerAATGAATTGCTTGACATCCATCA (SEQ ID NO: 5). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forhuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

To detect mouse Lysophospholipase I, a mouse Lysophospholipase Ispecific probe was prepared by PCR using the forward primerGGCTATGCCTTCTTGGTTTGATA (SEQ ID NO:11) and the reverse primerTGCCTGTTTAATTCCAGATTCATC (SEQ ID NO: 12). To normalize for variations inloading and transfer efficiency membranes were stripped and probed formouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUAN™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human Lysophospholipase I Expressionby Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human LysophospholipaseI RNA, using published sequences (GenBank accession number AF081281,incorporated herein as SEQ ID NO: 3, GenBank accession number AI567956,the complement of which is incorporated herein as SEQ ID NO: 17, andGenBank accession number AF052112, incorporated herein as SEQ ID NO:18). The oligonucleotides are shown in Table 1. Some of theseoligonucleotides also are complementary to mouse Lysophospholipase I RNA(GenBank accession numbers AI510294, A1573714 and AI663523, incorporatedherein as SEQ ID NOs: 19, 20 and 21, respectively) as indicated in Table2. “Target site” indicates the first (5′-most) nucleotide number on theparticular target sequence to which the oligonucleotide binds. Allcompounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human Lysophospholipase I mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom two experiments. If present, “N.D.” indicates “no data”.

TABLE 1 Inhibition of human Lysophospholipase I mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB NO120285 5′UTR 3 6 agctcacagcgcaagcggaa 55 22 120286 Coding 3 41gggttgacatgttattgccg 37 23 120287 Coding 3 81 gcggtggccttccgggcggc 49 24120288 Coding 3 114 gtatctcccaatccatgcag 77 25 120289 Coding 3 128cccatccgtgcccagtatct 39 26 120290 Coding 3 139 aaaggcttctgcccatccgt 2527 120291 Coding 3 141 gcaaaggcttctgcccatcc 56 28 120292 Coding 3 146tacctgcaaaggcttctgcc 56 29 120293 Coding 3 149 tgatacctgcaaaggcttct 6930 120294 Coding 3 262 tgaatctggtgaaagcccaa 72 31 120295 Coding 3 283aatcccagattcatcctcct 28 32 120296 Coding 3 311 aagcttttatattttctgct 6033 120297 Coding 3 318 tcaatcaaagcttttatatt 38 34 120298 Coding 3 328cacttcttgatcaatcaaag 72 35 120299 Coding 3 336 ccattcttcacttcttgatc 7136 120300 Coding 3 341 gaatgccattcttcacttct 71 37 120301 Coding 3 346agaaggaatgccattcttca 59 38 120302 Coding 3 351 ctgttagaaggaatgccatt 7039 120303 Coding 3 398 tatataaagataaagctcct 0 40 120304 Coding 3 426gccagtttctgctgtgtggt 64 41 12O305 Coding 3 447 caactgagtgcagtgacacc 7242 120306 Coding 3 452 gccagcaactgagtgcagtg 74 43 120307 Coding 3 457tggaagccagcaactgagtg 63 44 120308 Coding 3 462 cgaagtggaagccagcaact 7045 120309 Coding 3 467 aagcccgaagtggaagccag 61 46 120310 Coding 3 471aaggaagcccgaagtggaag 62 47 120311 Coding 3 533 aatccccgtggcactggaga 6348 120312 Coding 3 572 ccgtaagagaaccaaacatc 57 49 120313 Coding 3 599gattcaccaatgtttttagt 54 50 120314 Coding 3 620 ttttaaaggtcacattggct 6251 120315 Coding 3 656 cctgttgacacgaactgtgc 95 52 120316 Coding 3 672ttgacatccatcatttcctg 53 53 120317 Stop 3 708 caatcaattggaggtaggag 67 54Codon 120318 3′UTR 3 778 gcatgggaaaaggtttacac 75 55 120319 3′UTR 3 809cactgcaaaacattagaaat 49 56 120320 3′UTR 3 822 caaaacattttaacactgca 86 57120321 3′UTR 3 827 atttgcaaaacattttaaca 26 58 120322 3′UTR 3 839ttatcggcatgtatttgcaa 68 59 120323 3′UTR 3 844 ctgtgttatcggcatgtatt 68 60120324 3′UTR 3 863 tgaggagatattatttgatc 54 61 120325 3′UTR 3 867ctcatgaggagatattattt 66 62 120326 3′UTR 3 871 atttctcatgaggagatatt 71 63120327 3′UTR 3 882 aaagatcataaatttctcat 42 64 120328 3′UTR 3 903gaatacatgtatagaaactt 67 65 120329 3′UTR 3 934 ctaatatagtagatcctggg 81 66120330 3′UTR 3 1026 tttcaaatgatgtaataaaa 0 67 120331 3′UTR 3 1109actaagtcacagcatgcata 60 68 120332 3′UTR 3 1136 ctaagcaattttggaataaa 6 69120333 3′UTR 3 1140 gtgactaagcaattttggaa 54 70 120334 3′UTR 3 1159aaaatacagacactgcatgg 71 71 120335 3′UTR 3 1176 atatgaacacatatataaaa 2272 120336 3′UTR 3 1218 aataccacctcattcttatt 67 73 120337 3′UTR 3 1222atgtaataccacctcattct 80 74 120338 3′UTR 3 1261 tcttgacaataaacagcatt 7375 120339 3′UTR 3 1274 cgattttactttttcttgac 73 76 120340 3′UTR 3 1337gaaataatatcagggaaaat 0 77 120341 3′UTR 3 1480 atatctgtgacccagaatgt 68 78120342 3′UTR 3 1523 tgtctggtttctctataaag 75 79 120343 3′UTR 3 1604cctgggttgaagatcctaat 79 80 120344 3′UTR 3 1667 atatgatctaattattcttt 5581 120345 3′UTR 3 1738 atgagaagatgaaatcatct 66 82 120346 3′UTR 3 1754aagtaaactctatactatga 63 83 120347 3′UTR 3 1784 cttctacagtagttggtttc 7384 120348 3′UTR 3 1963 cataagtttgttttcaaata 17 85 120349 3′UTR 3 1966acccataagtttgttttcaa 72 86 120350 3′UTR 3 2032 ttttgctacatattaatctg 6687 128351 3′UTR 3 2O61 aacacagttgagaaatatca 58 88 120352 3′UTR 3 2134tatcaattagcacccattta 74 89 120353 3′UTR 3 2160 atagtccattactaaattat 6290 120354 3′UTR 3 2185 tatggcttcattattaacat 72 91 120355 3′UTR 3 2359atgaaaaacatttacacttt 18 92 120356 3′UTR 3 2371 ttgatagaaaccatgaaaaa 4493 120357 3′UTR 3 2389 attaaattttatttcacatt 18 94 120362 3′UTR 17 256aaaaagccaaaattaaattt 15 95 120358 5′UTR 18 27 gcgcccgcgcgtccagggtc 0 96120359 5′UTR 18 41 gctaccttccgcgcgcgccc 7 97 120360 5′UTR 18 86ccaccgggcgcacgctcagg 33 98 120361 5′UTR 18 125 gcggcccaagggcgtgcgag 1599

As shown in Table 1, SEQ ID NOs 22, 25, 28, 29, 30, 31, 33, 35, 36, 37,38, 39, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 57,59, 60, 61, 62, 63, 65, 66, 68, 70, 71, 73, 74, 75, 76, 78, 79, 80, 81,82, 83, 84, 86, 87, 88, 89, 90 and 91 demonstrated at least 50%inhibition of human Lysophospholipase I expression in this assay and aretherefore preferred.

Example 16 Antisense Inhibition of Mouse Lysophospholipase I Expressionby Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and aDeoxy Dap.

In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mouseLysophospholipase I RNA, using published sequences (GenBank accessionnumber U89352, incorporated herein as SEQ ID NO: 10, GenBank accessionnumber AI510294, incorporated herein as SEQ ID NO: 19, GenBank accessionnumber AI573714, incorporated herein as SEQ ID NO: 20, and GenBankaccession number AI663523, incorporated herein as SEQ ID NO: 21). Theoligonucleotides are shown in Table 2. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′0deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseLysophospholipase I mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”.

TABLE 2 Inhibition of mouse Lysophospholipase I mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB NO120287 Coding 21 83 gcggtggccttccgggcggc 67 24 120291 Coding 21 143gcaaaggcttctgcccatcc 95 28 120292 Coding 21 148 tacctgcaaaggcttctgcc 9529 120299 Coding 21 338 ccattcttcacttcttgatc 93 36 120300 Coding 21 343gaatgccattcttcacttct 96 37 120301 Coding 21 348 agaaggaatgccattcttca 9038 120302 Coding 21 353 ctgttagaaggaatgccatt 95 39 120304 Coding 21 428gccagtttctgctgtgtggt 81 41 120305 Coding 21 449 caactgagtgcagtgacacc 6942 120306 Coding 21 454 gccagcaactgagtgcagtg 70 43 120307 Coding 21 459tggaagccagcaactgagtg 82 44 120308 Coding 21 464 cgaagtggaagccagcaact 9445 120309 Coding 21 469 aagcccgaagtggaagccag 87 46 120316 Coding 20 41ttgacatccatcatttcctg 97 53 120317 Stop 20 77 caatcaattggaggtaggag 81 54Codon 120320 3′UTR 20 190 caaaacattttaacactgca 97 57 120321 3′UTR 20 195atttgcaaaacattttaaca 65 58 120330 3′UTR 20 388 tttcaaatgatgtaataaaa 5667 120334 3′UTR 20 507 aaaatacagacactgcatgg 94 71 120364 Coding 10 23cacaacggcgggcatcggag 90 100 120365 Coding 10 51 ccgcggcggtggccttccgg 89101 120366 Coding 10 78 tatctcccaatccgtgaagg 98 102 120367 Coding 10 82cctgtatctcccaatccgtg 98 103 120368 Coding 10 143 tggacagatgtatttgatgt 70104 120369 Coding 10 180 tattcatatttaatgtgact 78 105 120370 Coding 10185 agccatattcatatttaatg 90 106 120371 Coding 10 191aggcatagccatattcatat 91 107 120372 Coding 10 204 tatcaaaccaagaaggcata 90108 120373 Coding 10 209 aacgatatcaaaccaagaag 92 109 120374 Coding 10225 aatctggtgaaagtccaacg 95 110 120375 Coding 10 245tccagattcatcttcctggg 56 111 120376 Coding 10 260 tgctgcctgtttaattccag 89112 120377 Coding 10 280 atcaaggcttttacggtttc 86 113 120378 Coding 10313 ttagaaggaatgccattctt 90 114 120379 Coding 10 339gagaaaatcctcccaaaata 92 115 120380 Coding 10 447 gcccctgtgaaaacgaagcc 90116 120381 Coding 10 480 gaacggaaatatctcgatta 95 117 120382 Coding 16504 ggtcacaatctccatggcac 96 118 120383 Coding 10 510ctaaagggtcacaatctcca 97 119 120384 Coding 10 534 taagagaaccaaacattagg 91120 120385 Coding 10 561 ttatcaatgcttttagtctt 90 121 120386 Coding 10574 acattggctggatttatcaa 96 122 120387 Coding 10 626catttcctgctgacatgagc 94 123 120388 Coding 10 631 tccatcatttcctgctgaca 95124 120389 Coding 10 641 gtgcttgacatccatcattt 92 125 120390 Coding 10658 aggagcttatcaatgaagtg 93 126 120391 Coding 10 661ggtaggagcttatcaatgaa 94 127 l20419 3′UTR 19 455 agggccattaattgaagaat 91128 120420 3′UTR 19 499 gagaaaattacatagaagga 31 129 120421 3′UTR 19 551cagaaagccatattagtttt 48 130 120423 Stop 20 87 ttagtgatgtcaatcaattg 85133 Codon 120395 Stop 20 94 aggcctcttagtgatgtcaa 89 134 Codon 1203963′UTR 20 98 ctcaaggcctcttagtgatg 94 135 120397 3′UTR 20 141gaagaggtttatactctact 93 136 120398 3′UTR 20 153 ggtcagtcatgggaagaggt 94137 120399 3′UTR 20 163 gaagttctatggtcagtcat 95 138 120400 3′UTR 20 177cactgcacacattagaagtt 94 139 120401 3′UTR 20 202 ggtatgtatttgcaaaacat 90142 120402 3′UTR 20 214 gtctgtatcattggtatgta 91 143 120403 3′UTR 20 231gagggtaacatcatttagtc 90 144 120404 3′UTR 20 235 ccatgagggtaacatcattt 96145 120405 3′UTR 20 266 cgcatagacacttaaaagga 97 146 120406 3′UTR 20 287gaattgtataatacaaatat 64 147 120407 3′UTR 20 308 ctgtaatacttagtatattc 93148 120408 3′UTR 20 332 gaatctagttgcttcactta 96 149 120409 3′UTR 20 353ttttgctgaatttgagacaa 92 150 120410 3′UTR 20 355 tattttgctgaatttgagac 89151 120411 3′UTR 20 365 tttgttatcttattttgctg 92 152 120412 3′UTR 28 449acagaactaagaagtcactt 93 154 120413 3′UTR 20 487 agactgaacaattgtggagt 68155 120414 3′UTR 20 492 catggagactgaacaattgt 94 156 120415 3′UTR 20 512acagaaaaatacagacactg 96 158 120416 3′UTR 20 516 acaaacagaaaaatacagac 91159 120417 3′UTR 20 526 ctatgtgaatacaaacagaa 88 160 120418 3′UTR 20 530tcatctatgtgaatacaaac 86 161 120422 Start 21 29 ttgccgcacatccaccggct 90162 Codon 120393 Coding 21 43 gagcggacatgttgttgccg 74 163 120394 Coding21 47 atcggagcggacatgttgtt 64 164

As shown in Table 2, SEQ ID NOs 24, 28, 29, 36, 37, 38, 39, 41, 42, 43,44, 45, 46, 53, 54, 57, 58, 67, 71, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 133, 134, 135, 136, 137, 138,139, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155,156, 158, 159, 160, 161, 162, 163 and 164 demonstrated at least 50%inbition of mouse Lysophospholipase I expression in this experiment andare therefore preferred.

Example 17 Western Blot Analysis of Lysophospholipase I Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, supended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to Lysophospholipase Iis used, with a radiolabelled or fluorescently labeled secondaryantibody directed against the primary antibody species. Bands arevisualized using a PHOSPHORIMAGER™ (Molecular Dynamics, SunnyvaleCalif.).

164 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcgctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2atgcattctg cccccaagga 20 3 2417 DNA Homo sapiens CDS (36)...(728) 3cttccttccg cttgcgctgt gagctgaggc ggtgt atg tgc ggc aat aac atg 53 MetCys Gly Asn Asn Met 1 5 tca acc ccg ctg ccc gcc atc gtg ccc gcc gcc cggaag gcc acc gct 101 Ser Thr Pro Leu Pro Ala Ile Val Pro Ala Ala Arg LysAla Thr Ala 10 15 20 gcg gtg att ttc ctg cat gga ttg gga gat act ggg cacgga tgg gca 149 Ala Val Ile Phe Leu His Gly Leu Gly Asp Thr Gly His GlyTrp Ala 25 30 35 gaa gcc ttt gca ggt atc aga agt tca cat atc aaa tat atctgc ccg 197 Glu Ala Phe Ala Gly Ile Arg Ser Ser His Ile Lys Tyr Ile CysPro 40 45 50 cat gcg cct gtt agg cct gtt aca tta aat atg aac gtg gct atgcct 245 His Ala Pro Val Arg Pro Val Thr Leu Asn Met Asn Val Ala Met Pro55 60 65 70 tca tgg ttt gat att att ggg ctt tca cca gat tca cag gag gatgaa 293 Ser Trp Phe Asp Ile Ile Gly Leu Ser Pro Asp Ser Gln Glu Asp Glu75 80 85 tct ggg att aaa cag gca gca gaa aat ata aaa gct ttg att gat caa341 Ser Gly Ile Lys Gln Ala Ala Glu Asn Ile Lys Ala Leu Ile Asp Gln 9095 100 gaa gtg aag aat ggc att cct tct aac aga att att ttg gga ggg ttt389 Glu Val Lys Asn Gly Ile Pro Ser Asn Arg Ile Ile Leu Gly Gly Phe 105110 115 tct cag gga gga gct tta tct tta tat act gcc ctt acc aca cag cag437 Ser Gln Gly Gly Ala Leu Ser Leu Tyr Thr Ala Leu Thr Thr Gln Gln 120125 130 aaa ctg gca ggt gtc act gca ctc agt tgc tgg ctt cca ctt cgg gct485 Lys Leu Ala Gly Val Thr Ala Leu Ser Cys Trp Leu Pro Leu Arg Ala 135140 145 150 tcc ttt cca cag ggt cct atc ggt ggt gct aat aga gat att tctatt 533 Ser Phe Pro Gln Gly Pro Ile Gly Gly Ala Asn Arg Asp Ile Ser Ile155 160 165 ctc cag tgc cac ggg gat tgt gac cct ttg gtt ccc ctg atg tttggt 581 Leu Gln Cys His Gly Asp Cys Asp Pro Leu Val Pro Leu Met Phe Gly170 175 180 tct ctt acg gtg gaa aaa cta aaa aca ttg gtg aat cca gcc aatgtg 629 Ser Leu Thr Val Glu Lys Leu Lys Thr Leu Val Asn Pro Ala Asn Val185 190 195 acc ttt aaa acc tat gaa ggt atg atg cac agt tcg tgt caa caggaa 677 Thr Phe Lys Thr Tyr Glu Gly Met Met His Ser Ser Cys Gln Gln Glu200 205 210 atg atg gat gtc aag caa ttc att gat aaa ctc cta cct cca attgat 725 Met Met Asp Val Lys Gln Phe Ile Asp Lys Leu Leu Pro Pro Ile Asp215 220 225 230 tga cgtcactaag aggccttgtg tagaagtaca ccagcatcattgtagtagag 778 tgtaaacctt ttcccatgcc cagtcttcaa atttctaatg ttttgcagtgttaaaatgtt 838 ttgcaaatac atgccgataa cacagatcaa ataatatctc ctcatgagaaatttatgatc 898 ttttaagttt ctatacatgt attcttataa gacgacccag gatctactatattagaatag 958 atgaagcagg tagcttcttt tttctcaaat gtaattcagc aaaataatacagtactgcca 1018 ccagattttt tattacatca tttgaaaatt agcagtatgc ttaatgaaaatttgttcagg 1078 tataaatgag cagttaagat ataaacaatt tatgcatgct gtgacttagtctatggattt 1138 attccaaaat tgcttagtca ccatgcagtg tctgtatttt tatatatgtgttcatatata 1198 cataatgatt ataatacata ataagaatga ggtggtatta cattattcctaataataggg 1258 ataatgctgt ttattgtcaa gaaaaagtaa aatcgttctc ttcaattaatggccctttta 1318 ttttgggacc aggcttttat tttccctgat attatttcta tttaatactcttttctctca 1378 agaaaaaaaa aaaagtttgt tttttcttta ttgtccttca tagcaggccaagtattgcct 1438 ctctgcaata gacagctact gtcaatacat gctgtaattt gacattctgggtcacagata 1498 taaggtattt aaaatctatt tatgctttat agagaaacca gacattaaaacttcatgcac 1558 tacttatttc gaattactgt accttatcca aatttacacc tagctattaggatcttcaac 1618 ccaggtaaca ggaataattc tgtggtttca tttttctgta aacaactgaaagaataatta 1678 gatcatattc tagtatgttc tgaaatatct ttaagactga tcttaaaaactaacttctaa 1738 gatgatttca tcttctcata gtatagagtt tactttgtac acgttgaaaccaactactgt 1798 agaagatgag gaatctattg taattttttg ctttattttc atctgccagtggacttattt 1858 gaattttcac tttagtcaaa ttattttttg tattagtttt tgatgcagacataaaaatag 1918 caatcatttt aaattgtcaa aatttccaga ttactggtaa aaattatttgaaaacaaact 1978 tatgggtaat aaaggctagt cagaacccta taccataaag tgtagttaccatacagatta 2038 atatgtagca aaaatgtatg cttgatattt ctcaactgtg ttaatttttctgctgtattc 2098 cagctgacca aaacaatatt aagaatgcat ctttataaat gggtgctaattgataatgga 2158 aataatttag taatggacta tacaggatgt taataatgaa gccatatgtttatgtctgga 2218 tttaaaaatt ttaaacaatc atttactatg tcatttttct ttaccttgaagaacataaac 2278 tgttatttca cttctacaaa tcagcaagat attatttatg gcaagaaatattccattgaa 2338 atattgtgct gtaacatggg aaagtgtaaa tgtttttcat ggtttctatcaatgtgaaat 2398 aaaatttaat tctgaaaaa 2417 4 22 DNA Artificial SequencePCR Primer 4 tccagccaat gtgaccttta aa 22 5 23 DNA Artificial SequencePCR Primer 5 aatgaattgc ttgacatcca tca 23 6 32 DNA Artificial SequencePCR Probe 6 cctatgaagg tatgatgcac agttcgtgtc aa 32 7 19 DNA ArtificialSequence PCR Primer 7 gaaggtgaag gtcggagtc 19 8 20 DNA ArtificialSequence PCR Primer 8 gaagatggtg atgggatttc 20 9 20 DNA ArtificialSequence PCR Probe 9 caagcttccc gttctcagcc 20 10 693 DNA Mus musculusCDS (1)...(693) 10 atg tgc ggc aac aac atg tcc gct ccg atg ccc gcc gttgtg ccg gcc 48 Met Cys Gly Asn Asn Met Ser Ala Pro Met Pro Ala Val ValPro Ala 1 5 10 15 gcc cgg aag gcc acc gcc gcg gtt att ttc ctt cac ggattg gga gat 96 Ala Arg Lys Ala Thr Ala Ala Val Ile Phe Leu His Gly LeuGly Asp 20 25 30 aca ggg cat gga tgg gca gaa gcc ttt gca ggt atc aaa agtccc cac 144 Thr Gly His Gly Trp Ala Glu Ala Phe Ala Gly Ile Lys Ser ProHis 35 40 45 atc aaa tac atc tgt cca cat gcc cct gtg atg cca gtc aca ttaaat 192 Ile Lys Tyr Ile Cys Pro His Ala Pro Val Met Pro Val Thr Leu Asn50 55 60 atg aat atg gct atg cct tct tgg ttt gat atc gtt gga ctt tca cca240 Met Asn Met Ala Met Pro Ser Trp Phe Asp Ile Val Gly Leu Ser Pro 6570 75 80 gat tcc cag gaa gat gaa tct gga att aaa cag gca gca gaa acc gta288 Asp Ser Gln Glu Asp Glu Ser Gly Ile Lys Gln Ala Ala Glu Thr Val 8590 95 aaa gcc ttg ata gat caa gaa gtg aag aat ggc att cct tct aac agg336 Lys Ala Leu Ile Asp Gln Glu Val Lys Asn Gly Ile Pro Ser Asn Arg 100105 110 att att ttg gga gga ttt tct cag gga ggc gcc ttg tct tta tac act384 Ile Ile Leu Gly Gly Phe Ser Gln Gly Gly Ala Leu Ser Leu Tyr Thr 115120 125 gct ctc acc aca cag cag aaa ctg gct ggt gtc act gca ctc agt tgc432 Ala Leu Thr Thr Gln Gln Lys Leu Ala Gly Val Thr Ala Leu Ser Cys 130135 140 tgg ctt cca ctt cgg gct tcg ttt tca cag ggg ccg atc aac agt gct480 Trp Leu Pro Leu Arg Ala Ser Phe Ser Gln Gly Pro Ile Asn Ser Ala 145150 155 160 aat cga gat att tcc gtt ctc cag tgc cat gga gat tgt gac ccttta 528 Asn Arg Asp Ile Ser Val Leu Gln Cys His Gly Asp Cys Asp Pro Leu165 170 175 gtt ccc cta atg ttt ggt tct ctt act gtt gaa aga cta aaa gcattg 576 Val Pro Leu Met Phe Gly Ser Leu Thr Val Glu Arg Leu Lys Ala Leu180 185 190 ata aat cca gcc aat gta acc ttc aaa atc tat gaa ggc atg atgcac 624 Ile Asn Pro Ala Asn Val Thr Phe Lys Ile Tyr Glu Gly Met Met His195 200 205 agc tca tgt cag cag gaa atg atg gat gtc aag cac ttc att gataag 672 Ser Ser Cys Gln Gln Glu Met Met Asp Val Lys His Phe Ile Asp Lys210 215 220 ctc cta cct cca att gat tga 693 Leu Leu Pro Pro Ile Asp 225230 11 23 DNA Artificial Sequence PCR Primer 11 ggctatgcct tcttggtttgata 23 12 24 DNA Artificial Sequence PCR Primer 12 tgcctgttta attccagattcatc 24 13 26 DNA Artificial Sequence PCR Probe 13 cgttggactt tcaccagattcccagg 26 14 20 DNA Artificial Sequence PCR Primer 14 ggcaaattcaacggcacagt 20 15 20 DNA Artificial Sequence PCR Primer 15 gggtctcgctcctggaagct 20 16 27 DNA Artificial Sequence PCR Probe 16 aaggccgagaatgggaagct tgtcatc 27 17 326 DNA Homo sapiens unsure 99 unknown 17taattgataa tggaaataat ttagtaatgg gctatacagg atgttaataa tgaagccata 60tgtttatgtc tggatttaaa aattttaaac aatcatttnc tatgtcattt ttttttacct 120tgaagaacat aaactgttat ttccctttta caaatcagca agatattatt tatggcaaaa 180aatattccct ttaaatattg tgctgtaaca tgggaaagtg taaatgtttt tcatggtttc 240tatcaatgtg aaataaaatt taattttggc ttttttgtga aaaaaaaaaa aaaaaaaaaa 300aaaaaaaaaa aaaaaagggc ggccgc 326 18 1556 DNA Homo sapiens CDS(195)...(887) 18 cggcgggcga ggggcagggc agggcggacc ctggacgcgc gggcgcgcgcggaaggtagc 60 gcggggccgc gttggcgcgc acgcgcctga gcgtgcgccc ggtggggccggccgggactc 120 gccgctcgca cgcccttggg ccgcggccgg gcgcccgctc ttccttccgcttgcgctgtg 180 agctgaggcg gtgt atg tgc ggc aat aac atg tca acc ccg ctgccc gcc 230 Met Cys Gly Asn Asn Met Ser Thr Pro Leu Pro Ala 1 5 10 atcgtg ccc gcc gcc cgg aag gcc acc gct gcg gtg att ttc ctg cat 278 Ile ValPro Ala Ala Arg Lys Ala Thr Ala Ala Val Ile Phe Leu His 15 20 25 gga ttggga gat act ggg cac gga tgg gca gaa gcc ttt gca ggt atc 326 Gly Leu GlyAsp Thr Gly His Gly Trp Ala Glu Ala Phe Ala Gly Ile 30 35 40 aga agt tcacat atc aaa tat atc tgc ccg cat gcg cct gtt agg cct 374 Arg Ser Ser HisIle Lys Tyr Ile Cys Pro His Ala Pro Val Arg Pro 45 50 55 60 gtt aca ttaaat atg aac gtg gct atg cct tca tgg ttt gat att att 422 Val Thr Leu AsnMet Asn Val Ala Met Pro Ser Trp Phe Asp Ile Ile 65 70 75 ggg ctt tca ccagat tca cag gag gat gaa tct ggg att aaa cag gca 470 Gly Leu Ser Pro AspSer Gln Glu Asp Glu Ser Gly Ile Lys Gln Ala 80 85 90 gca gaa aat ata aaagct ttg att gat caa gaa gtg aag aat ggc att 518 Ala Glu Asn Ile Lys AlaLeu Ile Asp Gln Glu Val Lys Asn Gly Ile 95 100 105 cct tct aac aga attatt ttg gga ggg ttt tct cag gga gga gct tta 566 Pro Ser Asn Arg Ile IleLeu Gly Gly Phe Ser Gln Gly Gly Ala Leu 110 115 120 tct tta tat act gccctt acc aca cag cag aaa ctg gca ggt gtc act 614 Ser Leu Tyr Thr Ala LeuThr Thr Gln Gln Lys Leu Ala Gly Val Thr 125 130 135 140 gca ctc agt tgctgg ctt cca ctt cgg gct tcc ttt cca cag ggt cct 662 Ala Leu Ser Cys TrpLeu Pro Leu Arg Ala Ser Phe Pro Gln Gly Pro 145 150 155 atc ggt ggt gctaat aga gat att tct att ctc cag tgc cac ggg gat 710 Ile Gly Gly Ala AsnArg Asp Ile Ser Ile Leu Gln Cys His Gly Asp 160 165 170 tgt gac cct ttggtt ccc ctg atg ttt ggt tct ctt acg gtg gaa aaa 758 Cys Asp Pro Leu ValPro Leu Met Phe Gly Ser Leu Thr Val Glu Lys 175 180 185 cta aaa aca ttggtg aat cca gcc aat gtg acc ttt aaa acc tat gaa 806 Leu Lys Thr Leu ValAsn Pro Ala Asn Val Thr Phe Lys Thr Tyr Glu 190 195 200 ggt atg atg cacagt tcg tgt caa cag gaa atg atg gat gtc aag caa 854 Gly Met Met His SerSer Cys Gln Gln Glu Met Met Asp Val Lys Gln 205 210 215 220 ttc att gataaa ctc cta cct cca att gat tga cgtcactaag aggccttgtg 907 Phe Ile AspLys Leu Leu Pro Pro Ile Asp 225 230 tagaagtaca ccagcatcat tgtagtagagtgtaaacctt ttcccatgcc cagtcttcaa 967 atttctaatg ttttgcagtg ttaaaatgttttgcaaatac atgccaataa cacagatcaa 1027 ataatatctc ctcatgagaa atttatgatcttttaagttt ctatacatgt attcttataa 1087 gacgacccag gatctactat attagaatagatgaagcagg tagcttcttt tttctcaaat 1147 gtaattcagc aaaataatac agtactgccaccagattttt tattacatca tttgaaaatt 1207 agcagtatgc ttaatgaaaa tttgttcaggtataaatgag cagttaagat ataaacaatt 1267 tatgcatgct gtgacttagt ctatggatttattccaaaat tgcttagtca ccatgcagtg 1327 tctgtatttt tatatatgtg ttcatatatacataatgatt ataatacata ataagaatga 1387 ggtggtatta cattattcct aataatagggataatgctgt ttattgtcaa gaaaaagtaa 1447 aatcgttctc ttcaattaat ggcccttttattttgggacc aggcttttat tttccctgat 1507 attatttcta tttaatactc ttttctctcaaaaaaaaaaa aaaaaaaaa 1556 19 572 DNA Mus musculus unsure 482 unknown 19ttctaatgtg tgcagtgtta aaatgttttg caaatacata ccaatgatac agactaaatg 60atgttaccct catgggaaat atatgatcct tttaagtgtc tatgcatata tttgtattat 120acaattcaga atatactaag tattacagta ggtaagtgaa gcaactagat tctttgtctc 180aaattcagca aaataagata acaaacagtt ttattacatc atttgaaaat tactagtatg 240ttctgtgaag atgtgttcag gtaagatgta agtgacttct tagttctgtg agttagacta 300tagttttact ccacaattgt tcagtctcca tgcagtgtct gtatttttct gtttgtattc 360acatagatga tactataata gctaagaatc agatggtatt acattatccc taataatggg 420gataatgctt ttgagtgtca aaaagtaata ctgcattctt caattaatgg cccttctatt 480gngagaccag gctttttttc cttctatgta attttctcag aaaaaacagg gntctttttg 540attttccgag aaaactaata tggctttctg ca 572 20 608 DNA Mus musculus unsure598 unknown 20 ctt caa aat cta tga agg cat gat gca cag ctc atg tca gcagga aat 48 Leu Gln Asn Leu Arg His Asp Ala Gln Leu Met Ser Ala Gly Asn 15 10 15 gat gga tgt caa gca ctt cat tga taa gct cct acc tcc aat tga ttg96 Asp Gly Cys Gln Ala Leu His Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 acatcactaag aggccttgag tagaagttca ccagcatcac agtagtagag tataaacctc 157ttcccatgac tgaccataga acttctaatg tgtgcagtgt taaaatgttt tgcaaataca 217taccaatgat acagactaaa tgatgttacc ctcatgggaa atatatgatc cttttaagtg 277tctatgcgta tatttgtatt atacaattca gaatatacta agtattacag taggtaagtg 337aagcaactag attctttgtc tcaaattcag caaaataaga taacaaacag ttttattaca 397tcatttgaaa attactagta tgttctgtga agatgtgttc aggtaagatg taagtgactt 457cttagttctg tgagttagac tatagtttta ctccacaatt gttcagtctc catgcagtgt 517ctgtattttt ctgtttgtat tcacatagat gatactataa tagctaagaa tcagatggta 577ttacattatc cctaatagtg nggataatgc t 608 21 727 DNA Mus musculus unsure639 unknown 21 aaaaaaaatt tccgacgcac tgtccgccag ccggtgg atg tgc ggc aacaac atg 55 Met Cys Gly Asn Asn Met 1 5 tcc gct ccg atg ccc gcc gtt gtgccg gcc gcc cgg aag gcc acc gcc 103 Ser Ala Pro Met Pro Ala Val Val ProAla Ala Arg Lys Ala Thr Ala 10 15 20 gcg gtt att ttc ctt cac gga ttg ggagat aca ggg cat gga tgg gca 151 Ala Val Ile Phe Leu His Gly Leu Gly AspThr Gly His Gly Trp Ala 25 30 35 gaa gcc ttt gca ggt atc aaa agt ccc cacatc aaa tac atc tgt cca 199 Glu Ala Phe Ala Gly Ile Lys Ser Pro His IleLys Tyr Ile Cys Pro 40 45 50 cat gcc cct gtg atg cca gtc aca tta aat atgaat atg gct atg cct 247 His Ala Pro Val Met Pro Val Thr Leu Asn Met AsnMet Ala Met Pro 55 60 65 70 tct tgg ttt gat atc gtt gga ctt tca cca gattcc cag gaa gat gaa 295 Ser Trp Phe Asp Ile Val Gly Leu Ser Pro Asp SerGln Glu Asp Glu 75 80 85 tct gga att aaa cag gca gca gaa acc gta aaa gccttg ata gat caa 343 Ser Gly Ile Lys Gln Ala Ala Glu Thr Val Lys Ala LeuIle Asp Gln 90 95 100 gaa gtg aag aat ggc att cct tct aac agg att attttg gga gga ttt 391 Glu Val Lys Asn Gly Ile Pro Ser Asn Arg Ile Ile LeuGly Gly Phe 105 110 115 tct cag gga ggc gcc ttg tct tta tac act gct ctcacc aca cag cag 439 Ser Gln Gly Gly Ala Leu Ser Leu Tyr Thr Ala Leu ThrThr Gln Gln 120 125 130 aaa ctg gct ggt gtc act gca ctc agt tgc tgg cttcca ctt cgg gct 487 Lys Leu Ala Gly Val Thr Ala Leu Ser Cys Trp Leu ProLeu Arg Ala 135 140 145 150 tcg ttt tca cag ggg ccg atc aac agt gct aatcga gat att tcc gtt 535 Ser Phe Ser Gln Gly Pro Ile Asn Ser Ala Asn ArgAsp Ile Ser Val 155 160 165 ctc cag tgc cat gga gat tgt gac cct tta gttccc cta atg ttt ggt 583 Leu Gln Cys His Gly Asp Cys Asp Pro Leu Val ProLeu Met Phe Gly 170 175 180 tct ctt act gtt gaa aga cta aaa gca ttg ataaat cca gcc aat gta 631 Ser Leu Thr Val Glu Arg Leu Lys Ala Leu Ile AsnPro Ala Asn Val 185 190 195 acc ttc ana atc tat gaa ggc atg atg cac agctca tgt cag cag gaa 679 Thr Phe Xaa Ile Tyr Glu Gly Met Met His Ser SerCys Gln Gln Glu 200 205 210 atg ant gat gtc aag cac ttc att gat aag ctncta cct nca att gat 727 Met Xaa Asp Val Lys His Phe Ile Asp Lys Xaa LeuPro Xaa Ile Asp 215 220 225 230 22 20 DNA Artificial Sequence AntisenseOligonucleotide 22 agctcacagc gcaagcggaa 20 23 20 DNA ArtificialSequence Antisense Oligonucleotide 23 gggttgacat gttattgccg 20 24 20 DNAArtificial Sequence Antisense Oligonucleotide 24 gcggtggcct tccgggcggc20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 gtatctcccaatccatgcag 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26cccatccgtg cccagtatct 20 27 20 DNA Artificial Sequence AntisenseOligonucleotide 27 aaaggcttct gcccatccgt 20 28 20 DNA ArtificialSequence Antisense Oligonucleotide 28 gcaaaggctt ctgcccatcc 20 29 20 DNAArtificial Sequence Antisense Oligonucleotide 29 tacctgcaaa ggcttctgcc20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 tgatacctgcaaaggcttct 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31tgaatctggt gaaagcccaa 20 32 20 DNA Artificial Sequence AntisenseOligonucleotide 32 aatcccagat tcatcctcct 20 33 20 DNA ArtificialSequence Antisense Oligonucleotide 33 aagcttttat attttctgct 20 34 20 DNAArtificial Sequence Antisense Oligonucleotide 34 tcaatcaaag cttttatatt20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 cacttcttgatcaatcaaag 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36ccattcttca cttcttgatc 20 37 20 DNA Artificial Sequence AntisenseOligonucleotide 37 gaatgccatt cttcacttct 20 38 20 DNA ArtificialSequence Antisense Oligonucleotide 38 agaaggaatg ccattcttca 20 39 20 DNAArtificial Sequence Antisense Oligonucleotide 39 ctgttagaag gaatgccatt20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 tatataaagataaagctcct 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41gccagtttct gctgtgtggt 20 42 20 DNA Artificial Sequence AntisenseOligonucleotide 42 caactgagtg cagtgacacc 20 43 20 DNA ArtificialSequence Antisense Oligonucleotide 43 gccagcaact gagtgcagtg 20 44 20 DNAArtificial Sequence Antisense Oligonucleotide 44 tggaagccag caactgagtg20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 cgaagtggaagccagcaact 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46aagcccgaag tggaagccag 20 47 20 DNA Artificial Sequence AntisenseOligonucleotide 47 aaggaagccc gaagtggaag 20 48 20 DNA ArtificialSequence Antisense Oligonucleotide 48 aatccccgtg gcactggaga 20 49 20 DNAArtificial Sequence Antisense Oligonucleotide 49 ccgtaagaga accaaacatc20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 gattcaccaatgtttttagt 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51ttttaaaggt cacattggct 20 52 20 DNA Artificial Sequence AntisenseOligonucleotide 52 cctgttgaca cgaactgtgc 20 53 20 DNA ArtificialSequence Antisense Oligonucleotide 53 ttgacatcca tcatttcctg 20 54 20 DNAArtificial Sequence Antisense Oligonucleotide 54 caatcaattg gaggtaggag20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 gcatgggaaaaggtttacac 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56cactgcaaaa cattagaaat 20 57 20 DNA Artificial Sequence AntisenseOligonucleotide 57 caaaacattt taacactgca 20 58 20 DNA ArtificialSequence Antisense Oligonucleotide 58 atttgcaaaa cattttaaca 20 59 20 DNAArtificial Sequence Antisense Oligonucleotide 59 ttatcggcat gtatttgcaa20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 ctgtgttatcggcatgtatt 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61tgaggagata ttatttgatc 20 62 20 DNA Artificial Sequence AntisenseOligonucleotide 62 ctcatgagga gatattattt 20 63 20 DNA ArtificialSequence Antisense Oligonucleotide 63 atttctcatg aggagatatt 20 64 20 DNAArtificial Sequence Antisense Oligonucleotide 64 aaagatcata aatttctcat20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 gaatacatgtatagaaactt 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66ctaatatagt agatcctggg 20 67 20 DNA Artificial Sequence AntisenseOligonucleotide 67 tttcaaatga tgtaataaaa 20 68 20 DNA ArtificialSequence Antisense Oligonucleotide 68 actaagtcac agcatgcata 20 69 20 DNAArtificial Sequence Antisense Oligonucleotide 69 ctaagcaatt ttggaataaa20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 gtgactaagcaattttggaa 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71aaaatacaga cactgcatgg 20 72 20 DNA Artificial Sequence AntisenseOligonucleotide 72 atatgaacac atatataaaa 20 73 20 DNA ArtificialSequence Antisense Oligonucleotide 73 aataccacct cattcttatt 20 74 20 DNAArtificial Sequence Antisense Oligonucleotide 74 atgtaatacc acctcattct20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 tcttgacaataaacagcatt 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76cgattttact ttttcttgac 20 77 20 DNA Artificial Sequence AntisenseOligonucleotide 77 gaaataatat cagggaaaat 20 78 20 DNA ArtificialSequence Antisense Oligonucleotide 78 atatctgtga cccagaatgt 20 79 20 DNAArtificial Sequence Antisense Oligonucleotide 79 tgtctggttt ctctataaag20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 cctgggttgaagatcctaat 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81atatgatcta attattcttt 20 82 20 DNA Artificial Sequence AntisenseOligonucleotide 82 atgagaagat gaaatcatct 20 83 20 DNA ArtificialSequence Antisense Oligonucleotide 83 aagtaaactc tatactatga 20 84 20 DNAArtificial Sequence Antisense Oligonucleotide 84 cttctacagt agttggtttc20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 cataagtttgttttcaaata 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86acccataagt ttgttttcaa 20 87 20 DNA Artificial Sequence AntisenseOligonucleotide 87 ttttgctaca tattaatctg 20 88 20 DNA ArtificialSequence Antisense Oligonucleotide 88 aacacagttg agaaatatca 20 89 20 DNAArtificial Sequence Antisense Oligonucleotide 89 tatcaattag cacccattta20 90 20 DNA Artificial Sequence Antisense Oligonucleotide 90 atagtccattactaaattat 20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91tatggcttca ttattaacat 20 92 20 DNA Artificial Sequence AntisenseOligonucleotide 92 atgaaaaaca tttacacttt 20 93 20 DNA ArtificialSequence Antisense Oligonucleotide 93 ttgatagaaa ccatgaaaaa 20 94 20 DNAArtificial Sequence Antisense Oligonucleotide 94 attaaatttt atttcacatt20 95 20 DNA Artificial Sequence Antisense Oligonucleotide 95 aaaaagccaaaattaaattt 20 96 20 DNA Artificial Sequence Antisense Oligonucleotide 96gcgcccgcgc gtccagggtc 20 97 20 DNA Artificial Sequence AntisenseOligonucleotide 97 gctaccttcc gcgcgcgccc 20 98 20 DNA ArtificialSequence Antisense Oligonucleotide 98 ccaccgggcg cacgctcagg 20 99 20 DNAArtificial Sequence Antisense Oligonucleotide 99 gcggcccaag ggcgtgcgag20 100 20 DNA Artificial Sequence Antisense Oligonucleotide 100cacaacggcg ggcatcggag 20 101 20 DNA Artificial Sequence AntisenseOligonucleotide 101 ccgcggcggt ggccttccgg 20 102 20 DNA ArtificialSequence Antisense Oligonucleotide 102 tatctcccaa tccgtgaagg 20 103 20DNA Artificial Sequence Antisense Oligonucleotide 103 cctgtatctcccaatccgtg 20 104 20 DNA Artificial Sequence Antisense Oligonucleotide104 tggacagatg tatttgatgt 20 105 20 DNA Artificial Sequence AntisenseOligonucleotide 105 tattcatatt taatgtgact 20 106 20 DNA ArtificialSequence Antisense Oligonucleotide 106 agccatattc atatttaatg 20 107 20DNA Artificial Sequence Antisense Oligonucleotide 107 aggcatagccatattcatat 20 108 20 DNA Artificial Sequence Antisense Oligonucleotide108 tatcaaacca agaaggcata 20 109 20 DNA Artificial Sequence AntisenseOligonucleotide 109 aacgatatca aaccaagaag 20 110 20 DNA ArtificialSequence Antisense Oligonucleotide 110 aatctggtga aagtccaacg 20 111 20DNA Artificial Sequence Antisense Oligonucleotide 111 tccagattcatcttcctggg 20 112 20 DNA Artificial Sequence Antisense Oligonucleotide112 tgctgcctgt ttaattccag 20 113 20 DNA Artificial Sequence AntisenseOligonucleotide 113 atcaaggctt ttacggtttc 20 114 20 DNA ArtificialSequence Antisense Oligonucleotide 114 ttagaaggaa tgccattctt 20 115 20DNA Artificial Sequence Antisense Oligonucleotide 115 gagaaaatcctcccaaaata 20 116 20 DNA Artificial Sequence Antisense Oligonucleotide116 gcccctgtga aaacgaagcc 20 117 20 DNA Artificial Sequence AntisenseOligonucleotide 117 gaacggaaat atctcgatta 20 118 20 DNA ArtificialSequence Antisense Oligonucleotide 118 ggtcacaatc tccatggcac 20 119 20DNA Artificial Sequence Antisense Oligonucleotide 119 ctaaagggtcacaatctcca 20 120 20 DNA Artificial Sequence Antisense Oligonucleotide120 taagagaacc aaacattagg 20 121 20 DNA Artificial Sequence AntisenseOligonucleotide 121 ttatcaatgc ttttagtctt 20 122 20 DNA ArtificialSequence Antisense Oligonucleotide 122 acattggctg gatttatcaa 20 123 20DNA Artificial Sequence Antisense Oligonucleotide 123 catttcctgctgacatgagc 20 124 20 DNA Artificial Sequence Antisense Oligonucleotide124 tccatcattt cctgctgaca 20 125 20 DNA Artificial Sequence AntisenseOligonucleotide 125 gtgcttgaca tccatcattt 20 126 20 DNA ArtificialSequence Antisense Oligonucleotide 126 aggagcttat caatgaagtg 20 127 20DNA Artificial Sequence Antisense Oligonucleotide 127 ggtaggagcttatcaatgaa 20 128 20 DNA Artificial Sequence Antisense Oligonucleotide128 agggccatta attgaagaat 20 129 20 DNA Artificial Sequence AntisenseOligonucleotide 129 gagaaaatta catagaagga 20 130 20 DNA ArtificialSequence Antisense Oligonucleotide 130 cagaaagcca tattagtttt 20 131 131000 132 132 000 133 20 DNA Artificial Sequence Antisense Oligonucleotide133 ttagtgatgt caatcaattg 20 134 20 DNA Artificial Sequence AntisenseOligonucleotide 134 aggcctctta gtgatgtcaa 20 135 20 DNA ArtificialSequence Antisense Oligonucleotide 135 ctcaaggcct cttagtgatg 20 136 20DNA Artificial Sequence Antisense Oligonucleotide 136 gaagaggtttatactctact 20 137 20 DNA Artificial Sequence Antisense Oligonucleotide137 ggtcagtcat gggaagaggt 20 138 20 DNA Artificial Sequence AntisenseOligonucleotide 138 gaagttctat ggtcagtcat 20 139 20 DNA ArtificialSequence Antisense Oligonucleotide 139 cactgcacac attagaagtt 20 140 140000 141 141 000 142 20 DNA Artificial Sequence Antisense Oligonucleotide142 ggtatgtatt tgcaaaacat 20 143 20 DNA Artificial Sequence AntisenseOligonucleotide 143 gtctgtatca ttggtatgta 20 144 20 DNA ArtificialSequence Antisense Oligonucleotide 144 gagggtaaca tcatttagtc 20 145 20DNA Artificial Sequence Antisense Oligonucleotide 145 ccatgagggtaacatcattt 20 146 20 DNA Artificial Sequence Antisense Oligonucleotide146 cgcatagaca cttaaaagga 20 147 20 DNA Artificial Sequence AntisenseOligonucleotide 147 gaattgtata atacaaatat 20 148 20 DNA ArtificialSequence Antisense Oligonucleotide 148 ctgtaatact tagtatattc 20 149 20DNA Artificial Sequence Antisense Oligonucleotide 149 gaatctagttgcttcactta 20 150 20 DNA Artificial Sequence Antisense Oligonucleotide150 ttttgctgaa tttgagacaa 20 151 20 DNA Artificial Sequence AntisenseOligonucleotide 151 tattttgctg aatttgagac 20 152 20 DNA ArtificialSequence Antisense Oligonucleotide 152 tttgttatct tattttgctg 20 153 153000 154 20 DNA Artificial Sequence Antisense Oligonucleotide 154acagaactaa gaagtcactt 20 155 20 DNA Artificial Sequence AntisenseOligonucleotide 155 agactgaaca attgtggagt 20 156 20 DNA ArtificialSequence Antisense Oligonucleotide 156 catggagact gaacaattgt 20 157 157000 158 20 DNA Artificial Sequence Antisense Oligonucleotide 158acagaaaaat acagacactg 20 159 20 DNA Artificial Sequence AntisenseOligonucleotide 159 acaaacagaa aaatacagac 20 160 20 DNA ArtificialSequence Antisense Oligonucleotide 160 ctatgtgaat acaaacagaa 20 161 20DNA Artificial Sequence Antisense Oligonucleotide 161 tcatctatgtgaatacaaac 20 162 20 DNA Artificial Sequence Antisense Oligonucleotide162 ttgccgcaca tccaccggct 20 163 20 DNA Artificial Sequence AntisenseOligonucleotide 163 gagcggacat gttgttgccg 20 164 20 DNA ArtificialSequence Antisense Oligonucleotide 164 atcggagcgg acatgttgtt 20

What is claimed is:
 1. An antisense compound 8 to 30 nucleobases inlength targeted to nucleobases 6 through 25 of a 5′ untranslated region,nucleobases 41 through 60, nucleobases 81 through 100, nucleobases 114through 168, nucleobases 262 through 281, nucleobases 283 through 302,nucleobases 311 through 370, nucleobases 426 through 445, nucleobases447 through 490, nucleobases 533 through 552, nucleobases 572 through591, nucleobases 599 through 618, nucleobases 620 through 639,nucleobases 656 through 691 of a coding region, nucleobases 708 through727 of a stop codon, or nucleobases 778 through 797, nucleobases 809through 901, nucleobases 903 through 922, nucleobases 934 through 953,nucleobases 1109 through 1128, nucleobases 1140 through 1195,nucleobases 1218 through 1241, nucleobases 1261 through 1293,nucleobases 1480 through 1499, nucleobases 1523 through 1542,nucleobases 1604 through 1623, nucleobases 1667 through 1686,nucleobases 1738 through 1773, nucleobases 1784 through 1803,nucleobases 1966 through 1985, nucleobases 2032 through 2051,nucleobases 2061 through 2080, nucleobases 2134 through 2153,nucleobases 2160 through 2179, nucleobases 2185 through 2204,nucleobases 2371 through 2408 of a 3′-untranslated region of humanLysophospholipase I (SEQ ID NO: 3), wherein said antisense compoundspecifically hybridizes with one of said regions and inhibits theexpression of human Lysophospholipase I of SEQ ID NO:
 3. 2. Theantisense compound of claim 1 which is an antisense oligonucleotide. 3.The antisense compound of claim 2 wherein the antisense oligonucleotidecomprises at least one modified internucleoside linkage.
 4. Theantisense compound of claim 3 wherein the modified internucleosidelinkage is a phosphorothioate linkage.
 5. The antisense compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 6. The antisense compound of claim 5 wherein themodified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. Theantisense compound of claim 2 wherein the antisense oligonucleotidecomprises at least one modified nucleobase.
 8. The antisense compound ofclaim 7 wherein the modified nucleobase is a 5-methylcytosine.
 9. Theantisense compound of claim 2 wherein the antisense compound is achimeric oligonucleotide.
 10. A composition comprising the antisensecompound of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 11. The composition of claim 10 further comprising a colloidaldispersion system.
 12. The composition of claim 10 wherein the antisensecompound is an antisense oligonucleotide.
 13. A method of inhibiting theexpression of human Lysophospholipase I in cells or tissues comprisingcontacting said cells or tissues in vitro with the compound of claim 1so that expression of human Lysophospholipase I is inhibited.
 14. Anantisense compound up to 30 nucleobases in length comprising SEQ ID NO:22, 24, 25, 28, 29, 30, 31, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 57, 58, 59, 60, 61, 62, 63, 65,66, 67, 68, 70, 71, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 86, 87,88, 89, 90, 91, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 133, 134, 135, 136, 137, 138, 139, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 156, 158, 159, 160,161, 162, 163 or 164 which inhibits the expression of humanLysophospholipase I (SEQ ID NO: 3) or mouse Lysophospholipase I of SEQID NO: 10, 19, 20 or
 21. 15. The compound of claim 14 which is anantisense oligonucleotide.
 16. The compound of claim 15 wherein theantisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 17. The compound of claim 16 wherein themodified internucleoside linkage is a phosphorothioate linkage.
 18. Thecompound of claim 15 wherein the antisense oligonucleotide comprises atleast one modified sugar moiety.
 19. The compound of claim 18 whereinthe modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 20. Thecompound of claim 15 wherein the antisense oligonucleotide comprises atleast one modified nucleobase.
 21. The compound of claim 20 wherein themodified nucleobase is a 5-methylcytosine.
 22. The compound of claim 15wherein the antisense oligonucleotide is a chimeric oligonucleotide. 23.A composition comprising the compound of claim 14 and a pharmaceuticallyacceptable carrier or diluent.
 24. The composition of claim 23 furthercomprising a colloidal dispersion system.
 25. The composition of claim23 wherein the antisense compound is an antisense oligonucleotide.
 26. Amethod of inhibiting the expression of human Lysophospholipase I incells or tissues comprising contacting said cells or tissues in vitrowith the antisense compound of claim 14 so that expression of humanLysophospholipase I is inhibited.